Tumor lysate loaded particles

ABSTRACT

Dendritic cells containing tumor lysate loaded particles are prepared. The dendritic cells present tumor antigens to elicit the Major Histocompatibility Complex class I pathway and can be used as a vaccine to treat cancer, including ocular melanoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/019,025, filed Sep. 5, 2013, which claims priority from U.S.provisional application No. 61/697,498, filed Sep. 6, 2012.

BACKGROUND OF THE INVENTION

A tumor cell exists in part because it has selected for one or moremutations that allows it to partially or completely escape immunesurveillance in vivo.

In an attempt to elicit an immune response to a tumor cell, previousresearchers have used dendritic cells, which are professionalantigen-presenting cells, to present tumor antigens to the immunesystem. For example, dendritic cells pulsed with peptide or tumor lysatehave been used to vaccinate melanoma patients.

However, simply presenting tumor antigens to the immune system in theforegoing manner has not been effective because such antigens weremerely endocytosed by the dendritic cells and generally presentedthrough the Major Histocompatibility Complex (MHC) class II, whichelicits only helper T cells and does not provide a robust immuneresponse.

In contrast, presenting tumor antigens via the MHC class I pathwaycontributes to a more robust anti-tumor immunity by activating CD8+ Tcells. Previous researchers have attempted to present tumor antigensthrough the MHC class I pathway by using gene transfer methods. However,these methods have disadvantages, including (1) a limited ability toidentify all of the important tumor-specific antigens, (2) a limitedability to map the genes of specific tumor antigens, (3) only one or asmall number of known tumor antigen genes can be introduced into adendritic cell and (4) the methods are time-consuming and cumbersome.

SUMMARY OF THE INVENTION

Some embodiments relate to an isolated dendritic cell comprising aphagocytosed component consisting essentially of (i) a particle and (ii)a tumor lysate loaded within the particle. In specific embodiments, thetumor lysate is present in an amount from about 200 μg to about 500 μg.In specific embodiments, the tumor lysate is present in an amount ofabout 200 μg.

In some embodiments, the tumor lysate is a lysate selected from a cancerselected from the group consisting of breast cancer, small cell lungcancer, non-small cell lung cancer, glioma, medulloblastoma,neuroblastoma, Wilms tumors, rhabdomyosarcoma, osteosarcoma, livercancer, pancreatic cancer, melanoma, prostate cancer and ocularmelanoma.

In some embodiments, the particle is a yeast cell wall particle. In someembodiments, the dendritic cell is an immature cell that has beenisolated for no more than 8 days.

Another embodiment relates to a vaccine comprising the foregoingisolated dendritic cell.

Some embodiments relate to a method for producing an isolated dendriticcell containing a tumor lysate loaded particle comprising: (i) loadingthe tumor lysate into the particle to produce the tumor lysate loadedparticle; (ii) freeze-drying the tumor lysate loaded particle; and (iii)incubating the tumor lysate loaded particle with a dendritic cell,wherein the incubating causes the dendritic cell to phagocytose thetumor lysate loaded particle.

In specific embodiments, the foregoing method further comprises (a)resuspending the tumor lysate loaded particle in solution and (b)freeze-drying the resuspended solution before step (iii). In specificembodiments, the tumor lysate is produced by freezing and thawing thetumor. In specific embodiments, the foregoing method further comprisesrepeating the freezing and thawing steps. In specific embodiments, theforegoing method further comprises cryopreserving the tumor lysate at−150° C.

In specific embodiments, step (iii) comprises: (a) adding tumor lysateinto a yeast cell wall particle, (b) incubating the yeast cell wallparticle, (c) freeze-drying the yeast cell wall particle and (d) washingthe yeast cell wall, wherein steps (b)-(c) are repeated at least oncewith a step of adding water into the yeast cell particle before step (b)is repeated.

In specific embodiments, step (iii) comprises: (a) contacting the tumorlysate loaded particle and the dendritic cell at a ratio of about 100:1,(b) incubating the tumor lysate loaded particle with the dendritic cellfor 1 to 2 hours and (c) collecting the dendritic cell and washing thecell.

In a preferred embodiment, a small amount of one or more immune responseenhancing adjuvants is added to the tumor lysis buffer prior toincubating with a dendritic cell. The addition of one or more adjuvantsincreases the effects of the tumor lysate on the dendritic cell whiledramatically decreasing any systemic effects from such adjuvants.Commonly used adjuvants include but are not limited to proteins,peptides, nucleic acids and carbohydrates.

Exemplary adjuvants include but are not limited to monophosphoryl lipidA, GpC ologonucleotides (such as GpC DNA), Poly I:C, Poly ICLC, potentMEW II epitope peptides, beta glucan, and dendritic cell stimulatingcytokines such as IL-12. Suitable adjuvants are those molecules known tointeract with receptors on dendritic cells in order to activatedendritic cells and further stimulate a more robust generation of Tcells, such as CD4+ and CD8+ T cells.

In one embodiment, the amount of one or more immune response enhancingadjuvants is at least about 5 μg, at least about 10 μg, at least about15 μg, at least about 20 μg, at least about 25 μg, at least about 30 μg,at least about 35 μg, at least about 40 μg, at least about 45 μg, atleast about 50 μg, at least about 60 μg, at least about 70 μg, at leastabout 80 μg, at least about 80 μg, at least about 90 μg, or at leastabout 100 μg. In one embodiment, the amount of adjuvant representsbetween 1-10% of the total lysate amount. The amount of adjuvant issufficient to stimulate receptors, such as the toll-like receptor, onthe dendritic cell.

Some embodiments relate to a method for treating cancer, comprisingadministering a vaccine comprising the foregoing isolated dendriticcell. In specific embodiments, the cancer is selected from the groupconsisting of breast cancer, small cell lung cancer, non-small cell lungcancer, glioma, medulloblastoma, neuroblastoma, Wilms tumors,rhabdomyosarcoma, osteosarcoma, liver cancer, pancreatic cancer,melanoma, prostate cancer and ocular melanoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a process for producing dendritic cells.

FIG. 2 depicts a process for producing tumor lysate.

FIG. 3 depicts a process for producing yeast cell wall particles.

FIG. 4 depicts a process for loading tumor lysate into yeast cell wallparticles.

FIG. 5 depicts a process for producing tumor lysate particle loadeddendritic cells.

FIG. 6 is a graph comparing the effect of tumor lysate particle loadeddendritic cells versus antigen pulsing of dendritic cells on B3Z cells.

FIG. 7A shows the lungs of control mice afflicted with B16F0 murinemelanoma.

FIG. 7B shows the lungs of mice afflicted with B16F0 murine melanoma buttreated with tumor lysate particle loaded dendritic cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference is made herein to various methodologies known to those ofordinary skill in the art. Publications and other materials settingforth such known methodologies to which reference is made areincorporated herein by reference in their entirety as though set forthin full.

As used herein, the singular forms “a,” “an,” and “the” designate boththe singular and the plural, unless expressly stated to designate thesingular only. Likewise, singular forms of terms designate both thesingular and plural, unless expressly stated to designate the singularonly. For example, “vaccine” means “vaccine” or “vaccines.”

The term “about” in connection with numerical values and ranges meansthat the number comprehended is not limited to the exact number setforth herein, and is intended to refer to ranges substantially withinthe quoted range while not departing from the scope of the invention. Asused herein, “about” will be understood by persons of ordinary skill inthe art and will vary to some extent on the context in which it is used.

As used herein “subject” or “patient” denotes any animal in need oftreatment with a vaccine. For example, a subject may be suffering fromor at risk of developing a condition that can be treated or preventedwith a vaccine. As used herein “subject” or “patient” includes humans.

As used herein, the phrases “therapeutically effective amount” and“therapeutic level” mean that vaccine dosage or plasma concentration ina subject, respectively, that provides the specific response for whichthe vaccine is administered in a subject in need of such treatment. Forconvenience only, exemplary dosages, vaccine delivery amounts,therapeutically effective amounts and therapeutic levels are providedbelow with reference to adult human subject. Those skilled in the artcan adjust such amounts in accordance with standard practices as neededto treat a specific subject and/or condition/disease.

Tumor Lysate

As described herein, “tumor lysate” refers to a tumor that has beenlysed. Tumor lysis can occur under a number of conditions, includingrepeated freezing and thawing of the tumor, physical breakage of thetumor by homogenizing, contact with a hyper- or hypo-tonic solution andcontact with one or more non-ionic detergents. The tumor lysate is notcross-linked during the lysing process. In another embodiment, the tumorlysate is produced by mincing, grinding or mashing the tumor, orotherwise pulverizing the tumor using any known technique in the art. Inanother embodiment, the tumor lysate is produced by mincing, grinding,mashing or pulverizing the tumor in the presence of phosphate buffersolution (PBS), such as 1X PBS.

In specific embodiments, the tumor lysate is produced from a solid tumorweighing a minimum of 200 to 500 μg.

In another embodiment, the tumor lysate is produced by mincing,grinding, mashing or pulverizing the tumor followed by repeated freezingand thawing. In specific embodiments, the minced tumor is frozen andthawed multiple times. In specific embodiments, the minced tumor isfrozen and thawed at least 1, 2, 3 or 4 times. In some embodiments, thefreezing is performed in liquid nitrogen, and can be performed for 20minutes. In specific embodiments, the thawing is performed at roomtemperature. In another embodiment, the tumor lysate is stored at atemperature of about −135° C. or below after the freezing and thawingprocess. In specific embodiments, the tumor lysate is store at atemperature of −150° C. or below after the freezing and thawing process.

The tumor lysate can be prepared from any solid tumor including, but notlimited to carcinomas, and sarcomas. In some embodiments, the solidtumors are from tumors relating to breast cancer, small cell lungcancer, non-small cell lung cancer, glioma, medulloblastoma,neuroblastoma, Wilms tumors, rhabdomyosarcoma, osteosarcoma, livercancer, pancreatic cancer, melanoma, prostate cancer and ocularmelanoma.

A number of immune response enhancing agents can be added to the tumorlysis buffer as adjuvants to boost immune response such that when thetumor lysate is incubated with a dendritic cell, the adjuvants exhibitan increased effect on the dendritic cell while dramatically decreasingany systemic effects from such adjuvants.

It is within the purview of one of ordinary skill in the art to selectone or more suitable adjuvants for this invention. For instance,monophosphoryl lipid A, GpC oligonucleotides, Poly I:C, Poly ICLC,potent MHC II epitope peptides, and dendritic cell stimulating cytokinessuch as IL-12, IL-2, and GM-CSF are good adjuvant candidates of thisinvention.

Particle

As described herein, “particle” refers to any hollow and porousstructure that can contain tumor lysate therein and also allow thelysate to exit the structure. In some embodiments, the size of theparticle is about 0.5 to about 5 μm, which approximates the size ofbacterium to allow the particle to be ingested by monocytes, such asdendritic cells. In specific embodiments, the size of the particle isabout 0.5 to about 1 μm. In specific embodiments, the size of theparticle is about 0.5 to about 2.5 μm. In some embodiments, the particlecan be any particle with a glycan network, so long as the particle isabout 0.5 to about 5 μm in size.

In some embodiments, the particle is a bead vector. The bead vector isnot limited by shape or material, but can be any shape, size or materialthat allows the bead vector to be phagocytosed by monocytes, includingdendritic cells.

In another embodiment, the particle is a yeast cell wall particle(“YCWP”). The YCWP is prepared from yeast cell wall such that theparticle is porous and can contain lysate therein. In one embodiment,the YCWP is prepared from Saccharomyces cerevisiae. In anotherembodiment, the YCWP is a zymosan particle. In another embodiment, theYCWP approximates the size of microbial structures that cells of themononuclear phagocyte system and other phagocytic cells typicallyingests. In specific embodiments, the YCWP is about 1-5 μm.

In one embodiment, the YCWP is prepared by (a) suspending yeast toproduce a suspension, (b) incubating the suspension, (c) centrifugingthe suspension and removing the supernatant and (d) recovering theresulting YCWP. In another embodiment, steps (a)-(d) are repeated atleast 1, 2, 3 or 4 times.

In another embodiment, the YCWP is prepared by (a) suspending yeast in asolution to produce a first suspension, (b) incubating the firstsuspension, (c) centrifuging the first suspension and removing thesupernatant, (d) suspending the resulting pellet to produce a secondsuspension, (e) incubating the second suspension, (f) centrifuging thesecond suspension and removing the supernatant and (g) washing theresulting pellet to recover the YCWP. In another embodiment, the YCWP issterilized.

In specific embodiments, the yeast is suspended in NaOH, including 1MNaOH. In specific embodiments, the first suspension is incubated atabout 80° C. for about 1 hour or for 1 hour. In specific embodiments,the centrifuging is performed at about 2000 times gravity for about 10minutes, or at 2000 times gravity for 10 minutes. In specificembodiments, the pellet is suspended in water, including water at aboutpH 4.5 or at pH 4.5. In specific embodiments, the second suspension isincubated at about 55° C. for about 1 hour or at 55° C. for 1 hour. Inspecific embodiments, the pellet is washed in water at least 1, 2, 3 or4 times. In specific embodiments, the pellet is washed once.

In another embodiment, the YCWP is sterilized using isopropanol and/oracetone following washing of the pellet. In specific embodiments, otherknown alcohols are appropriate. In specific embodiments, the YCWP isallowed to fully dry after sterilization. In another embodiment, theYCWP is resuspended after being allowed to dry. In specific embodiments,the YCWP is resuspended in PBS, such as 1X PBS. In another embodiment,the YCWP is allowed to dry and then frozen before the tumor lysate isloaded into the YCWP, in order to place it in storage before use. Inspecific embodiments, the YCWP is freeze dried and store at about 4° C.or lower. In specific embodiments, the YCWP is freeze dried and store at4° C.

Tumor Lysate Loaded Particle

The particle is loaded with tumor lysate. In one embodiment, the tumorlysate is loaded into the particle by incubating the lysate and asuspension of particles together and allowing the lysate to penetrateinto the hollow insides of the particles.

In another embodiment, after the particle is loaded with tumor lysate,the combination is freeze-dried to create an anhydrous tumor lysatewithin the particle. By freeze-drying the particle loaded with tumorlysate, the lysate is trapped within the particle and ready to bephagocytosed by a monocyte, such as a dendritic cell. In specificembodiments, the freeze-drying is the only mechanism used to trap thelysate within the particle. In specific embodiments, the entrapment isnot caused by a separate component blocking the lysate from exiting theparticle, for example, by physical entrapment, hydrophobic binding, anyother binding. In specific embodiments, the entrapment is not caused bycrosslinking or otherwise attaching the lysate to the particle outsideof any attachment that may occur upon freeze-drying.

In another embodiment, the particle is resuspended in solution after thefreeze-drying. In specific embodiments, the solution is water. Inspecific embodiments, the particle is resuspended to allow additionaltumor lysate to penetrate the particle and then the combination isfreeze-dried again. In other embodiments, the combination is subjectedto multiple freeze-drying and resuspensions. In other embodiments, thetumor lysate loaded particle is sterilized in ethanol after thefreeze-drying and before use.

In specific embodiments, the tumor lysate is loaded into the particle by(a) incubating the lysate and a suspension of the particles, allowingthe lysate to penetrate into the hollow insides of the particles andfreeze-drying the suspension of particles loaded with lysate and (b)optionally resuspending the particles, incubating the resuspendedparticles and freeze drying the resuspended particles and any tumorlysate not already in the particle.

In specific embodiments using YCWPs, the number of YCWPs is about 1×10⁹and the volume of tumor lysate is about 50 μL (generated from about 200μg of tumor tissue). In specific embodiments, the number of YCWPs is1×10⁹ and the volume of tumor lysate is 50 μL (from about 200 μg oftumor tissue). In specific embodiments, the incubation in step (a) isfor about 2 hours at about 4° C. In specific embodiments, the incubationin step (a) is for 2 hours at 4° C. In some embodiments, the foregoingsuspension is freeze dried in step (b) over a period of about 2 hours orover a period of 2 hours. In some embodiments, the YCWPs in step (c) areresuspended in water, including about 50 μL of water or 50 μL of water.In some embodiments, the resuspended YCWPs are incubated in step (d) forabout 2 hours at about 4° C. or for 2 hours at 4° C.

Dendritic Cell

As described herein, “dendritic cell” refers to a cell generated from aperipheral blood mononuclear cell (“PBMC”). In one embodiment, adendritic cell is prepared by (a) collecting blood, (b) diluting theblood, (c) performing a density gradient separation of PBMCs, (d) lysingred blood cells and washing the PBMCs, (e) incubating the PBMCs, (f)removing nonadherent cells and (g) culturing adherent cells in media.

In some embodiments, the dendritic cell is an immature dendritic cellthat has been cultured for no more than 5 days. In other embodiments,the dendritic cell has been cultured for 6-8 days.

In specific embodiments, the blood is heparinized. In specificembodiments, the density gradient separation at step (c) comprisesplacing the blood in a Lymphocyte Separation Medium and thencentrifuging the blood. In specific embodiments, the centrifuging isperformed at about 1000 times gravity for about 20 minutes or at 1000times gravity for 20 minutes. In specific embodiments, a secondcentrifuging is performed before step (d) and is performed at about 500g for about 5 minutes or is performed at 500 g for 5 minutes. Inspecific embodiments, a third centrifuging is performed before step (d)and is performed at about 500 g for about 10 minutes or is performed at500 g for 10 minutes. In specific embodiments, the lysing is performedusing an ACK lysing solution, followed by incubation, preferably at roomtemperature for about 5 minutes, and followed by a subsequentcentrifugation. In specific embodiments, the PBMCs are washed inRPMI-1640 medium. In specific embodiments, the PBMCs are incubated atstep (e) in flasks at about 37° C. for about 1-2 hours or at 37° C. for1-2 hours. In specific embodiments, serum-free DC media is added to theflask.

In some embodiments, one or more cytokines is present in the culturemedia, including, but not limited to, granulocyte macrophage colonystimulating factor (800 units/ml) and IL-4 (500 units/ml).

Tumor Lysate Loaded Particles Phagocytosed in Dendritic Cells

The tumor lysate loaded particle is phagocytosed within a monocyte,preferably a dendrite cell. In one embodiment, the tumor lysate loadedparticle is incubated with a dendritic cell such that the cellphagocytoses the tumor lysate loaded particle.

In specific embodiments, the particle is incubated with the dendriticcell at a ratio of about 100:1 or at a ratio of 100:1. The incubationcan be performed for in about 1 hour, 1 hour or preferably less than 1hour.

In specific embodiments, the incubated dendritic cell containing thetumor lysate particle is collected and washed, for example, at least 1,2, 3 or 4 times. In other embodiments, the dendritic cells are incubatedafter washing and resuspended in freezing medium. In specificembodiments, the resuspension produces a concentration of about 10×10⁶cells per ml or 10×10⁶ cells per ml. In specific embodiments, theresuspension is frozen for storage before use.

Vaccine

In one embodiment, the dendritic cell containing a tumor lysate loadedparticle is used as a vaccine to prevent and/or treat a disease,including cancer. The disease to be treated is not particularlylimiting, but depends on the particular tumor lysate loaded into theparticle. For example, a vaccine using tumor lysate from a breast cancertumor is used to treat breast cancer. In another embodiment, a patient'sown tumor cells are used to create the vaccine. For example, the vaccinecan be produced using tumor lysate from a tumor associated with breastcancer and then administered to the breast cancer patient from which thetumor was extracted. In another embodiment, about 200 μL of a 10×10⁶concentration of dendritic cells containing tumor lysate loadedparticles forms one dose of the vaccine.

In another embodiment, the dose is administered by diluting the 200 μLaliquot to a final volume of 1 ml before administering the dose to apatient. In specific embodiments, the aliquot is diluted with sterilesaline containing 5% human serum albumin. In specific embodiments, the200 μL aliquot will need to be thawed before dilution. In such ascenario, the length of time between thawing and administration of thedose to a patient will be no longer than 2 hours. In some embodiments,the diluted aliquot is administered in a 3 cc syringe. In someembodiments, a syringe needle no smaller than 23 gauge is used.

In another embodiment, a patient is administered at least 1, 2, 3 or 4doses. In specific embodiments, a patient is re-vaccinated once every 4weeks. In specific embodiments, about 1-2 million dendritic cellscontaining tumor lysate loaded particles will be administered at eachvaccination. In another embodiment, the dendritic cells containing tumorlysate loaded particles are administered to a patient by injection. Inspecific embodiments, the tumor lysate loaded particles are injected ina patient at or near (1) a tumor or (2) a lymph node.

In some embodiments, the vaccine is not administered with any otherimmunosuppressive treatment, such as steroids or chemotherapy. Thevaccine can be administered using any technique, including intravenousinjection and inhalation.

The vaccine can also contain biological adjuvants, including but notlimited to nucleic acids such as GpC oligonucleotides, proteins orpeptide epitopes such as the tetanus toxoid MHC class II-binding p30peptide.

EXAMPLE 1 Preparing Dendritic Cells

Dendritic cells were generated from a patient's PBMCs. PBMCs werecollected from the patient by a blood draw of 200 ml following standardoperating procedures. The blood was then transferred to 250 mlcentrifuge tubes and diluted 1:1 with 1×PBS. Then, 35 ml of the dilutedblood was layered over 15 ml of room temperature Lymphocyte SeparationMedium (LSM; Mediatech) in 50 ml tubes and centrifuged at 1000 g for 20minutes at room temperature. The PBMC layers were removed by pipettingfrom the LSM gradients and placed into clean 50 ml centrifuge tubes.Four volumes of 1×PBS were added and the tubes were inverted to mix thecontents. The PBMCs were then centrifuged at 500 g at room temperaturefor 5 minutes. Ten ml of 1×PBS were added into each tube and the cellswere resuspended and pooled into 1 tube. The PBMCs were againcentrifuged at 500 g at room temperature for 10 minutes, resuspended in20 to 40 ml of ACK lysing solution (Cambrex) and incubated at roomtemperature for 5 minutes. The cells were then centrifuged again at 1500rpm for 5 minutes. The PBMCs were resuspended in 30 ml RPMI-1640 medium(Mediatech). The cells were then transferred into 2-4 T75 flasks. Theflasks were incubated at 37° C. for 1 to 2 hours. The non-adherent cellswere then removed by rinsing. Afterwards, 10 ml of 1×PBS were added intoeach flask, the flask swirled, and the PBS removed. Afterwards, 10 ml ofcomplete DC media (serum-free DC Medium+800 U/ml GM-CSF+1000 U/ml IL-4)was added to each flask. The flasks were then incubated at 37° C., 5%CO2 for 2 days. On Day 3, 10 ml of complete DC medium was added intoeach flask. The cells were then incubated for another 2 days. On Day 6or 7, the resulting immature DCs were ready for use.

FIG. 1 provides an overview of the generation of dendritic cells.

EXAMPLE 2 Preparing Tumor Lysate

A tumor sample was obtained from a patient. After separating fat andnecrotic tissue away from the tumor tissue, the tissue was weighed and1X PBS added (50 μL of PBS per 200 μg of tissue) and the tumor wasminced thoroughly with scalpels in 1X PBS. The tumor cells were thensubjected to 4 cycles of freeze and thaw. The freezing was performed inliquid nitrogen for 20 minutes and the thawing was performed at roomtemperature. Prepared tumor lysate was quantified by aspectrophotometer. An aliquot was taken for quality control testing. Theremainder was stored at ≤−135° C. in preparation for vaccinepreparation. Small amounts of adjuvant can optionally be added after thefreeze thaw cycles.

FIG. 2 provides an overview of the tumor cell lysate processing.

EXAMPLE 3 Preparing YCWP

YCWPs were prepared from Fleishmans Baker's Yeast or equivalent.Briefly, 10 g of Fleishmans Baker's yeast was suspended in 100 ml of 1 MNaOH and heated to 80° C. for one hour. The undissolved yeast cell wallswere recovered by centrifugation at 2000×g for 10 minutes. The recoveredyeast cell walls were then resuspended in 100 ml of water with the pHadjusted to 4.5 with HCl and incubated at 55° C. for an additional hour,and subsequently recovered by centrifugation. The recovered YCWPs werethen washed with water once, isopropanol 4 times and finally acetone 2times. Once the YCWPs were fully dried they were resuspended in PBS,counted, aliquoted into groups of 1×10⁹ particles and freeze dried foruse in manufacturing the vaccine.

FIG. 3 provides an overview of the yeast cell wall particles processing.

EXAMPLE 4 Preparing YCWP

Three grams of active dry yeast (Fleischmann's or equivalent) werewashed three times in water by suspending the yeast in 30 mL of sterilewater, vortexing, and centrifuging at 800-1000 x g for 5 minutes at roomtemperature. After decanting the supernatant, the yeast pellet wasresuspended in 50 mL of 1 M NaOH and heated in a 90° C. water bath for 1hour.

The yeast suspension was subsequently centrifuged at 800-1000 x g for 5minutes, and the pellet was resuspended in 25-30 mL of acid water (pHadjusted to 4.5 with HCl). The acid water wash step was repeated untilthe pH of the suspension is <7.0. Then the pellet was resuspended in 30mL acid water and incubated in a 75° C. water bath for 1 hour. The yeastpellet was recovered by centrifugation at 1000×g for 5 minutes, and thenwashed with 10 mL of sterile water 3 times, 10 mL of isopropanol 4 timesand finally 10 mL of acetone 2 times. The acetone was carefully removed,and the pellet was spread evenly on the glass surface of a beaker,allowed to air dry overnight.

The dried YWCPs were collected and stored in a vacuum jar at 4° C. andthen washed in 10-15 mL of filtered 70% ethanol 3 times. The YWCPs werebriefly sonicated on the final wash, and the sonication was repeated ifnecessary to disperse clumps. Once the ethanol was removed, the YWCPswere washed in sterile water. Aliquots of 100₁1.1 of YWCPs weredispensed into 2.0 mL rounded bottom snap top centrifuge tubes, placedin freezer for 1 hour, freeze dried, and stored in a vacuum jar at 4° C.for future use.

EXAMPLE 5 Loading Tumor Lysate into YCWP

A suspension of fully anhydrous YCWPs (1×10⁹) was placed in contact with50 of tumor lysate in PBS (from 200 μgs of tumor tissue) over a periodof 2 hours at 4° C., allowing the lysate to penetrate into the hollowinsides of the YCWPs to produce loaded YCWPs. The suspension was thenfreeze dried for 2 hours. After freeze drying, 50 μL of water was addedto the loaded YCWPs, incubated for another 2 hours at 4° C. and againfreeze dried to yield YCWPs with dry tumor lysate within their hollowinsides. The loaded YCWPs were then sterilized by washing in ethanol andmaintained in ethanol.

FIG. 4 provides an overview of the YCWPs loading procedure.

EXAMPLE 6 Loading YCWPs with Tumor Lysate

A patient tumor biopsy sample was mixed carefully with 50-100 μlof lysisbuffer (PBS) (depending on the amount of the tumor sample), avoidingbubbles during mixing, and was then incubated at 4° C. for 30 minutes.The mixture was subjected to freeze-thaw 3 times in acetone-dry ice bathand 37° C. water bath, and centrifuged at 4° C. for 10 minutes atmaximum speed. 50 μl of the prepared tumor lysate was added in a sterile2 mL centrifuge tube containing 10 mg of dried YCWPs such that theliquid tumor lysate covered the YCWPs. The mixture was incubated at 4°C. for 2 hours until the liquid tumor lysate soaked into the YCWPs.

The tube was then placed into a −85° C. freezer for 30 minutes for aquick freeze of the pellet. The tube was placed on freeze drierovernight. 50 μl of sterile water was added onto the dried yeast pelletand incubated at 4° C. for 2 hours to allow the liquid to soak into thepellets.

The tube was placed into a −85° C. freezer for 30 minutes for a quickfreeze of the pellet. The tube was then placed on freeze drierovernight. The dried particles were then resuspended in 1 mL of 70%ethanol and stored at 4° C. for future use.

EXAMPLE 8 Preparing Dendritic Cells Containing Tumor Lysate LoadedParticles

The tumor lysate loaded YCWPs in 70% ethanol suspension was centrifuged.The ethanol was removed carefully and replaced with 1 mL of PBS. Theloaded YCWPs were sonicated. The loaded YCWPs were washed with sterile1X PBS. After final wash, the loaded YCWPs were resuspended in PBS toapproximately 1×10⁸ particles/100 μl PBS.

The loaded YCWPs were added to a dendritic cell culture at a ratio of1:100, and the culture was returned to 37° C. incubator. Subsequently,the following factors were added to the culture: 50 μg/mL of TNF-α insterile water was added to the culture at a ratio of 1:5000 in volume (2μL per 10 mL of culture); 10 μg/mL of IL-β in sterile water was added tothe culture at a ratio of 1:1000 in volume; 10 μg/mL of IL-6 in sterilewater was added to the culture at a ratio of 1:1000 in volume; and 1mg/mL of PGE2 in 100% ethanol was added to the culture at a ratio of1:1000 in volume. After all factors were added and mixed into theculture, the culture was incubated overnight.

EXAMPLE 9 Harvest of Dendritic Cells, Preparation and Cryopresrvation ofVaccine

The dendritic cell culture prepared according to Example 9 was removedfrom the incubator. The following procedure was performed in a hoodunder sterile conditions. 10 mL of media were removed from cultureflask. The culture flask was rinsed with 4.0-4.5 mL of 1×PBS and alsoadded to the media.

1.5-2.0 mL of CellStripper™ was added to the culture flask. The cultureflask was placed in 37° C. incubator for 10-20 minutes. About 4 mL ofthe culture media were added back to the flask from the tube to wash andremove cells. The flask was washed to harvest as many cells as possible.The cells were counted on hemacytometer or Cellometer™. The supernatantwas removed after centrifugation.

Subsequently, the cells were resuspended in CryoStor™ 10 at 5×10⁶cells/mL, aliquoted into cryovials properly labeled with patient IDNumber, date and cell concentration at 1.25×10⁶ cells/mL per vial (about250 μL). A 250-500 μL portion was saved in a cryovial for sterilitytesting, and the remaining vials were stored in Styrofoam containers andplaced under −86° C. to step down freeze.

EXAMPLE 10 Preparation of the Solid Tumor Vaccine for PatientAdministration

One cryovial of patient's cell was removed from cryostorage andcarefully thawed at 37° C. in a water bath. Under sterile conditions, 1mL of sterile saline for injection with 5% human serum albumin (or 1 mLof sterile 1X PBS) was gently added to the cryovial containing thecells. After the cells were carefully resuspended, the entire volumefrom the cryovial was drawn and the syringe containing the tumor cellswas used for administration of the vaccine to a patient.

EXAMPLE 11 Dendritic Cells Containing Tumor Lysate Loaded ParticlesVersus Antigen Pulsed Dendritic Cells (DCs)

B3Z cells are a T cell hybridoma expressing a T-cell receptor thatspecifically recognizes the OVA₍₂₅₇₋₂₆₄₎ (SIINFEKL) epitope in thecontext of H-2K^(b) and carry a beta-galactosidase (1acZ) constructdriven by a nuclear factor of activated T cells elements from theinterleukin 2 promoter (X). These B3Z cells were used to evaluate theeffectiveness of dendritic cells pulsed with ovalbumin versus thoseloaded with ovalbumin by way of ovalbumin loaded YCWPs for antigenpresentation, resulting in a CD8+ T cell response.

Upon activation by interaction with MHC class I molecules on dendriticcells presenting ovalbumin epitopes, the B3Z cells were engineered torespond by expressing β-galactosidase. β-Galactosidase catalyzes abreakdown of X-gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside) toyield 5-bromo-4-chloro-3-hydroxyindole, a blue colored product.Spectroscopic measurement of this blue color gives a measure of theeffective MHC class I presentation of the ovalbumin epitope. The resultsof this experiment, shown in FIG. 6, demonstrate that loading dendriticcells by ovalbumin loaded YCWPs gave more than a 100 fold increase inCD8+ T cell response over dendritic cells pulsed with ovalbumin.

EXAMPLE 12 In Vitro Data

Dendritic cells were prepared from cells obtained from the bone marrowof the femur and tibia of both hind legs of a female C57BL/6J mouse.B16F0 murine melanoma cells were obtained (ATCC (CRL-6322)) and culturedusing standard tissue culture techniques. The dendritic cells wereloaded with YCWPs containing B16F0 tumor lysate (around 2×10⁻¹⁵ g/YCWP)at a ratio of 100:1 particles:DC by adding the particles at day 7 of adendritic cell culture for a period of 2 hours. Three days prior to thepreparation of the dendritic cells containing tumor lysate loadedparticles, female C57BL/6J mice were challenged with 0.75×10⁶ B16F0melanoma cells in 0.4 ml 1X PBS by intravenous injection. Once thedendritic cells containing tumor lysate loaded particles were prepared,each mouse in the treatment group was injected intravenously with 2×10⁶of dendritic cells containing tumor lysate loaded particles and thisvaccination was repeated for three weekly doses. The mice were monitoredup to four weeks for pulmonary metastasis.

At the end of four weeks (when one of the control mice died), the micewere sacrificed and any occurrence of metastases was counted. All fourcontrol animals that were not treated with dendritic cells containingtumor lysate loaded particles had more than 50 tumors. However, none ofthe treated animals had measurable metastases. This data indicates thatdendritic cells containing tumor lysate loaded particles is effective intreating cancer in a proven animal model system. The data are compiledin Table 1.

TABLE 1 Number of Metastases in Control and Treated Mice Number ofMetastases Group (including tumors at non-lung sites) Control: A >50B >50 C >50 D >50 Treated: A 0 B 0 C 0 D 0

Moreover, FIG. 7A shows the lungs of three of the control mice (onemouse died prior to the end of the experiment and the lungs were notable to be photographed) in this experiment and FIG. 7B shows the lungsof the four treated mice.

EXAMPLE 13 In Vivo Procedure

To vaccinate a subject, a dose of 1.25 million dendritic cellscontaining tumor lysate loaded particles can be cryopreserved in 0.2 mLof a serum-free, 10% dimethyl sulfoxide freezing medium (CryoStor™CS-10, BioLife Solutinos, Inc.). Before injection, the dendritic cellscan be thawed and diluted to a 1 mL with sterile saline for injectioncontaining 5% human serum albumin (Albuminar -25, Aventis Behring). Thedilution can then be transferred to a 3.0 cc syringe for injection andusing a needle no smaller than 23 gauge, which should be administeredwithin 2 hours of the thawing. The injection can be administeredsubcutaneously into an area of lymph nodes.

EXAMPLE 14 Isolation of Mononuclear Cells from Whole Peripheral BloodUsing the SepMate-50 System Procedure:

STEP PROCEDURE/WORK INSTRUCTIONS 1 Add 15 mL of lymphocyte separationmedium (LSM) to each SepMate tube by carefully, yet quickly, pipettingit through the central hole of each tube insert. 2 Pool the whole blood.3 Dilute the whole blood sample with twice the initial blood volume of1xPBS. 4 Add 30 mL diluted blood to the Sepmate tubes. 5 Centrifuge for10-15 minutes at room temperature. 6 Pour off the top layer containingthe enriched PBMC's from each Sepmate tube into new centrifuge tubes. 7Cap tubes and centrifuge 5 minutes. 8 Resuspend pellet in each tube withup to 1.0 mL ACK lysing buffer with pipette. Repeat cycles of adding 1XPBS to resuspend pellet and centrifuge. 9 Decant the supernatant, andaliquot 50 × 10⁶ cells suspended in 15 mL RPMI media per flask. Place inthe CO₂ incubator at 37° C. for 1-1.5 hours. 10 Remove flasks fromincubator. Wash pellet with 1X PBS. 11 add 15 mL complete DC Media(containing IL-4, GM-CSF, and Gentamycin) and place in the 37° CO₂incubator for 22-24 hours. 12 On Day 2, continue with inoculation ofcultures with YWCP's and cytokines after the dendritic cells haveincubated at 37° C. for approx. 22 hours.

EXAMPLE 15 Generation of Dendritic Cells Combined with Loaded YCWP's

Following the procedure in Example 14, the following methods areperformed:

I. Addition of YCWP's

STEP PROCEDURE/WORK INSTRUCTIONS 1 To each dendritic cell culture flask,add a sufficient volume of tumor lysate infused YCWP's at a ratio of1:100. (50-200 ul) and incubate for 1-2 hours.

II. Preparation And Addition of Cytokines

STEP PROCEDURE/WORK INSTRUCTIONS 1 Add TNF-α, 1β, IL-6 and PGE2 to eachculture flask,

EXAMPLE 16 Harvest of Cells, Preparation and Cryopreservation of Vaccine

Following the procedure in Example 15, the following methods areperformed:

Harvest of cells:

STEP PROCEDURE/WORK INSTRUCTIONS 1 Add 4.0-4.5 mL 1xPBS to each flaskcontaining 10 mL media. 2 Add 1.5-2.0 mL CellStripper ™ to each flask torelease mature cells and incubate at 37 C. 3 Centrifuge and emovesupernatant by decanting until ‘dry’ pellet remains.

II. Preparation of Vaccine and Cryopreservation:

STEP PROCEDURE/WORK INSTRUCTIONS 1 Resuspend cells in CryoStor ™ 10,aliquot and step down freeze.

1-15. (canceled)
 16. A method of treating cancer, comprisingadministering to a subject with cancer a composition comprising adendritic cell that has phagocytosed a yeast cell wall particle loadedwith a tumor lysate, wherein the composition comprises about 1-2 milliondendritic cells.
 17. The method of claim 16, wherein the dendritic cellis an immature cell that has been isolated for no more than 8 days. 18.The method of claim 16, wherein the yeast cell wall particle is loadedat a concentration of about 200 μg to about 500 μg of tumor lysate perabout 10⁹ yeast cell wall particles.
 19. The method of claim 16, whereinthe yeast cell wall particle is about 0.5 μm to about 5 μm in size. 20.The method of claim 16, wherein the tumor lysate is a lysate of a cancerselected from the group consisting of breast cancer, small cell lungcancer, non-small cell lung cancer, glioma, medulloblastoma,neuroblastoma, Wilms tumors, rhabdomyosarcoma, osteosarcoma, livercancer, pancreatic cancer, melanoma, prostate cancer and ocularmelanoma.
 21. The method of claim 16, wherein the composition furthercomprises a GpC oligonucleotide.
 22. The method of claim 16, wherein thecomposition further comprises a tetanus toxoid MHC class II-binding p30peptide.
 23. The method of claim 16, wherein the composition isadministered via an injection.
 24. The method of claim 23, wherein theinjection is an intravenous or subcutaneous injection.
 25. The method ofclaim 16, wherein the cancer is selected from the group consisting ofbreast cancer, small cell lung cancer, non-small cell lung cancer,glioma, medulloblastoma, neuroblastoma, Wilms tumors, rhabdomyosarcoma,osteosarcoma, liver cancer, pancreatic cancer, melanoma, prostate cancerand ocular melanoma.
 26. The method of claim 16, wherein the yeast cellwall particles were loaded with the tumor lysate by (i) contacting theyeast cell wall particle with the tumor lysate; and (ii) freeze dryingthe tumor lysate and the yeast cell wall particle to produce the tumorlysate loaded yeast cell wall particle.